There is considerable interest in determining the diagnostic value of stool specimens for detecting gastrointestinal disease caused by Microsporidia organisms, and a variety of staining modalities have been evaluated for their ability to visualize microsporidia in stool preparations. Unfortunately, the various species of microsporidia that infect humans can be identified accurately only by visualizing characteristic subcellular morphologic features of organisms in electron micrographs of tissue specimens. Because the efficacy of treatment regimens currently in development may be somewhat species-specific, and because invasive procedures are necessary to obtain appropriate specimens for electron microscopy (EM), a more widely available approach to identification than EM is required. We have published a report which describes our polymerase chain reaction (PCR) assay for amplification of microsporidial rRNA genes followed by restriction endonuclease digestion of PCR products as a method for identifying microsporidia in fresh stool. We are continuing to modify the assay to allow detection of microsporidia in formalin-fixed stool specimens. A Southern blot format using PCR-amplified DNA and species-specific DNA probes has been developed for use in microsporidial speciation. The final stage of the project has been to determine the sensitivity of our assay and the length of time the specimens can be stored in formalin and still allow detection of the microsporidia. We have demonstrated that prolonged storage of microsporidia in formalin or polyvinyl alcohol reduces the chances of amplification by PCR. We suggest washing stool specimens after fixation in formalin and storage of the washed specimen in phosphate-buffered saline. We have developed a shorter, cheaper, and simpler method for extraction of microsporidial DNA from stool.